Two-dimensional liquid chromatographic analyzer and analytical method

ABSTRACT

A two-dimensional liquid chromatographic analyzer, in which a temperature control part changes a temperature in a single-step manner and switches the temperature at a high speed. Also, the temperature control part has a holder around a separation column so that the temperature of the separation column can be changed to a preset temperature. A first separation column thereof changes elution time(s) of the objective component(s) by temperature modulation and the mobile phase of the first separation column is an aqueous mobile phase having a constant composition.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to Japanese PatentApplication Nos. 2015-080384 filed Mar. 25, 2015 and 2016—filed Mar. 25,2016. The entire contents of both of applications are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The disclosure relates to a liquid chromatograph. Specifically, thedisclosure relates to a two-dimensional liquid chromatographic analyzerhaving a plurality of separation columns and an analytical method.

2. Description of the Related Art

A liquid chromatograph is an apparatus configured to separate objectivecomponents in a sample by a separation column while feeding a mobilephase and to detect the components that flow out in an order of theirseparation by a detector such as a spectrophotometer so as to analyzethe components of the sample.

For the liquid chromatograph, separation columns of various separationmodes such as an ion-exchange mode, a normal phase mode, and a reversedphase mode are employed for separating ionic components, hydrophiliccomponents, hydrophobic components, and the like in samples and the kindof the mobile phase varies depending on the separation mode.

For the mobile phase, for improving separation and shortening analysistime, there is generally used gradient elution in which two or morekinds of mobile phases are fed while changing a mixing ratio thereof.

On the other hand, there is also known a method of achieving animprovement in separation and shortening of analysis time by changingthe temperature of the separation column. For example, in a reversedphase chromatograph in which, as a representative reversed phaseseparation column, an octadecylsilyl (ODS) group is used as a functionalgroup, it is generally known that elution is fastened when thetemperature of the separation column becomes high.

Japanese Patent No. 5362943 reports a simple and convenient drugmetabolizing ability evaluation system in which a filling agent whosesurface is covered with a polymer having hydration force that changeswithin the temperature range of 0 to 80° C. is used for a separationcolumn and objective components are separated and measured bycontrolling the temperature of the separation column.

However, in the case where components in a biological sample such asserum are analyzed using a liquid chromatograph, a plurality ofcomponents are present as a mixture in the biological sample, and use ofone column may result in insufficient separation or may take a longperiod of time for analysis.

In Japanese Patent No. 5362943, after the biological sample ispre-treated using a column for solid-phase extraction, objectivecomponents are analyzed through a separation column.

As a specific separation method, there is used a two-dimensional liquidchromatographic analytical method in which, using a flow channelswitching valve, columns of two kinds of different separation modes arecombined and the objective components separated by a first(first-dimensional) separation column are fractionated and thenintroduced into a second (second-dimensional) column to further separatethem.

In the two-dimensional liquid chromatography, it is common to performanalysis using an ion exchange column as the first-dimensional columnand a reversed phase column as the second-dimensional one.

In the first-dimensional ion exchange column, the objective componentsare separated by salt concentration or pH of a buffer solution as amobile phase and the separated objective components are introduced intothe second-dimensional reversed phase column. In the second-dimensionalreversed phase column, further separation is performed using a mobilephase containing an organic solvent.

JP-A-2008-96455 describes examples of the two-dimensional liquidchromatographic analyzer in which the first-dimensional separation andthe second-dimensional separation are performed.

The technique described in JP-A-2008-96455 is that, in order to performreplacement of the mobile phase in the system efficiently, thecomponents separated in the first dimension are once trapped andconcentrated by a trap column and the components trapped by the trapcolumn are further separated and analyzed in the second dimension. As aconfiguration thereof, there has been reported a two-dimensional liquidchromatograph capable of analyzing even any components using fewestthree trap columns.

JP-A-2008-96455 describes a two-dimensional liquid chromatographcomprising a first-dimensional separation flow channel for introducing asample injected from a sample injection part into a separation columnwith a mobile phase for separation to separate the sample, three trapcolumns, a flow channel of a mobile phase for analysis for supplying themobile phase for analysis, a second-dimensional analytical flow channelfor introducing the components trapped in the trap columns into ananalytical column with the mobile phase for analysis to analyze thecomponents, a flow channel of a mobile phase for desalination forsupplying the mobile phase for desalination, and a flow channelswitching mechanism for connecting the first-dimensional separation flowchannel to one trap column, connecting the flow channel of the mobilephase for desalination to another one trap column, connecting thefirst-dimensional separation flow channel to still another one trapcolumn, and also switching the connections of the trap columns and theflow channels, wherein the flow channel switching mechanism includes afirst and second two-position valves to which one end and another end ofeach of the three trap columns are connected, a third two-position valveto which the first-dimensional separation flow channel, the flow channelof the mobile phase for desalination, and the flow channel of the mobilephase for analysis are connected and whose one end is connected toanother end of three flow channels that are connected to the firsttwo-position valve, and a fourth two-position valve whose one end isconnected to another end of three flow channels that are connected tothe second two-position valve and the second-dimensional analytical flowchannel.

The flow channels are connected by valves so that the three trap columnscan independently take action. By connecting the flow channels so as toperform concentration in one trap column, desalination in another onetrap column, and elution in still another one trap columnsimultaneously, the concentration action, the desalination action, andthe elution action simultaneously proceed in respective different trapcolumns by valves, so that it is possible to continue second-dimensionalanalysis with three trap columns without limiting the number ofcomponents.

SUMMARY OF THE INVENTION

In a common two-dimensional liquid chromatograph, a buffer solution isused as a mobile phase of a first-dimensional ion exchange column.

In the ion exchange column, a buffer solution is used as a mobile phaseand objective components are separated by increasing ionic strength(salt concentration) of the buffer solution or changing pH thereof.Therefore, in the ion exchange column, there is used gradient elution inwhich two or more kinds of mobile phases are fed while changing a mixingratio thereof.

A salt is precipitated by mixing the buffer solution and the organicsolvent contained in the mobile phase of the second-dimensional reversedphase column and thus clogging is formed in a piping or thesecond-dimensional reversed phase column. In order to prevent theclogging, a trap column for feeding a mobile phase for desalinationbecomes necessary and there is a problem that system configuration iscomplicated by providing a switching valve for the trap column and thelike.

The disclosure provides a two-dimensional liquid chromatographicanalyzer and an analytical method, which are capable of performing highsensitive analysis of objective component(s) in a sample such as abiological one with a simple configuration in which a system for feedinga mobile phase for desalination is not needed by preventing the saltprecipitation.

The two-dimensional liquid chromatograph of the invention may beconfigured as follows.

A two-dimensional liquid chromatographic analyzer comprising: a liquidfeed part configured to feed a mobile phase; an injection partconfigured to inject a sample; a first separation column configured toseparate and fractionate the sample containing a plurality ofcomponents; a second separation column configured to separate thefractionated components; a temperature control part configured tocontrol temperature of each separation column; and a detection partconfigured to detect the separated components, wherein the temperaturecontrol part comprises a holder made of metal and configured to hold thefirst separation column so as to transmit a temperature change of thefirst temperature control part to the first separation column.

The first separation column may be a temperature-responsible gelmodification column having a functional polymer layer whose surfacenature is reversibly changed between hydrophilic nature and hydrophobicnature by temperature modulation or a reversed phase column to which itis possible to feed a 100% aqueous mobile phase, and the secondseparation column may be a reversed phase column.

In a two-dimensional liquid chromatographic analytical method, a mobilephase to be fed to a first separation column is an aqueous mobile phase,and the composition of the mobile phase to be fed to the firstseparation column is not changed at the separation and fractionation ofobjective components.

When the present analyzer is used, it becomes possible to analyzecomponents of a biological sample or the like with a simple system.

By adopting a temperature-responsible gel modification separation columnas a first-dimensional first separation column, it is possible toseparate major contaminant components and a drug group in a biologicalsample such as serum with an aqueous mobile phase alone.

The temperature-responsible gel modification separation column cancontrol elution time of a drug in the first-dimensional first separationcolumn by temperature control. By using the temperature-responsible gelmodification separation column, at the time when a plurality of drugsare simultaneously analyzed, the elution time of the drug group can becontrolled by temperature modulation and thus any drug can be introducedinto a second-dimensional second separation column.

The mobile phase of the first-dimensional first separation column is anaqueous mobile phase having a constant composition and it is notnecessary to use a buffer solution from which a salt may be precipitatedthrough mixing with a mobile phase of the second separation columncontaining an organic solvent at the introduction into thesecond-dimensional column. Therefore, it is unnecessary to feed a mobilephase for desalination and a trap column and a switching valve becomeunnecessary, so that system configuration becomes simple. The simplesystem configuration also improves operability and maintainability.

Next, the aqueous mobile phase is introduced into the second-dimensionalsecond separation column together with objective component(s). Thesecond separation column is a reversed phase column and the objectivecomponent(s) does not immediately elute from the second-dimensionalsecond separation column and remain in the second separation column.

For the mobile phase of the second separation column, gradient elutionwith an organic solvent and an aqueous mobile phase is used.

The objective component(s) remaining in the second separation column isonce converged in the column and is eluted with the organic solventcontained in the mobile phase of the second separation column, so thatthe peak(s) of the eluted objective component(s) is sharply detected.

Thereby, in the second-dimensional chromatogram, the peak(s) of theobjective component(s) becomes sharp and thus high sensitivity analysisis possible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an outline block diagram of a liquid chromatographicanalyzer;

FIG. 1B is a structural drawing of a temperature control part;

FIG. 2 is a graph showing a relationship between temperature preset andelution time;

FIG. 3 is a chromatogram showing a concentration effect by a liquidchromatographic analyzer;

FIGS. 4A and 4B are chromatograms obtained by analyzing drugs in serumby a liquid chromatographic analyzer, in which FIG. 4A shows afirst-dimensional chromatogram and FIG. 4B shows a second-dimensionalchromatogram; and

FIG. 5 shows temperature curves when being changed from one settemperature to another set temperature.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following will describe embodiments of the invention with referenceto the drawings.

FIG. 1A is an outline block diagram of a two-dimensional liquidchromatographic analyzer that is the first embodiment of the invention.

The two-dimensional liquid chromatographic analyzer comprises pumps 1and 2 for feeding eluents, a sample injection device 5 for injecting asample into the system, separation columns 3 and 4 for separating thesample sent to the system, temperature control parts 6 and 7 forcontrolling temperatures of separation columns, detectors 8 and 9 fordetecting sample components separated by the separation columns 3 and 4,and a valve 10 for connecting/separating individual flow channels.

In the two-dimensional liquid chromatographic analyzer, a pump 1 feedsan eluent A 11 a from an eluent container 11 to the sample injectiondevice 5. The sample injection device 5 injects a sample into a flowchannel together with the eluent A 11 a from the pump 1.

The components in the sample injected into the flow channel are sent tothe separation column (first separation column) 3 and are eluted into aflow channel 21 in an ascending order of interaction with the separationcolumn 3.

The separation columns 3 and 4 are controlled to preset temperaturessuitable for separation by the temperature control parts 6 and 7,respectively.

The components in the sample eluted into the flow channel 21 are sent tothe detector 8 through a six-way valve 10 and a flow channel 22 andcontaminant components alone are discharged from a flow channel 26 as awaste solution. When the separation of the contaminant components iscompleted, the flow channel 21 from the separation column 3 is connectedto a flow channel 23 though the six-way valve 10. At this time, eluentsB (12 a and 13 a) are fed from eluent containers 12 and 13 to the flowchannel 23 by a pump 2 through a flow channel 24 and the six-way valve10, and are fed to the separation column (second separation column) 4together with the eluent A 11 a containing the sample from theseparation column 3.

The components separated by the separation column 4 are eluted in anascending order of interaction with the separation column 4 andmeasurement of each component is performed on a detector 9.

The timing of switching the path of the flow channel 21 to the flowchannel 22 to the path of the flow channel 21 to the flow channel 23 bythe six-way valve 10 is decided through determination of the elutiontime of the contaminant components previously and, at measurement, theswitching of the flow channels is set based on the time.

The eluents B (12 a and 13 a) are fed by the pump 2 but, during timeperiods other than the time period where the flow channel 21 and theflow channel 23 are connected for feeding the sample solution from theseparation column 3 to the separation column 4, are discharged as awaste solution while the flow channel 24 and a flow channel 25 areconnected by the six-way valve 10.

The separation columns 3 and 4 are placed in the temperature controlparts 6 and 7, respectively. The temperature control part 6 of theseparation column 3 is configured to switch a temperature of theseparation column 3 to a target temperature. The temperature controlpart 6 has a holder 6 a around the separation column 3 so as to holdthat the separation column 3. The holder 6 a is made of metal such asaluminum alloy, copper alloy, etc., excellent in thermal conductivity.As shown by FIG. 1B, an inner structure of the holder 6 a is formed tofit an outer structure of the separation column 3, so that the holder 6a is close contactable with an outer circumference of the separationcolumn 3 so as to surround the outer circumference of the separationcolumn 3. According thereto, temperature change of the temperaturecontrol part 6 can be efficiently transmitted to the separation column3.

The temperature control part 6 is configured to change the temperatureof the separation column 3 from a first temperature to a secondtemperature in a single-step manner (e.g., in a steep manner) and switchthe temperature of the separation column 3 at a high speed to reach anew target temperature.

As the separation column 3, there is employed a temperature-responsiblegel modification column having a polymer layer of apolyN-isopropylacrylamide (PNIPAAm) layer and butyl methacrylate (BMA)as a hydrophobic comonomer introduced into the PNIPAAm chains onsurfaces of silica gel beads using a gel modification method.

On the surface of the polymer layer of the temperature-responsible gelmodification column of the separation column 3, a change betweenhydrophilic nature and hydrophobic nature occurs through a change in thestructure of the polymer chain on the surface caused by temperaturechange.

In the separation column 3, by means of the temperature control part 6,the hydrophilic and hydrophobic characteristics change through thechange in the structure of the polymer chain on the polymer layersurface of the temperature-responsible gel modification column, and theseparation column 3 is set at a temperature suitable for the separationof the objective component(s) by the interaction with the eluent.

The temperature control part 6 is provided with a heat block 6 b as aheat source. Temperature of the heat block 6 b is controlled by aPeltier element. The temperature control part 6 is configured to controlthe temperature of the separation column 3 by directly transmitting thetemperature of the heat block 6 b to the holder 6 a so as to change thetemperature of the separation column 3 to reach the set temperaturewithin a short time.

In a related-art liquid chromatograph, temperature change when beingswitched from one set temperature to new set temperature, thetemperature increases as shown by a curved line B in FIG. 5, so thattransient becomes long. In such a related-art liquid chromatograph, inorder to maintain reproducibility of a retention time, it becomesnecessary to perform the analysis after waiting for several times untilthe temperature becomes stable at the new set temperature. On the otherhand, the temperature control part 6 can change the temperature steeplyas shown by a curved line A in FIG. 5, so that transient can be madeshort and temperature can become stable at the new set temperaturequickly. The temperature control part 6 can change the temperature froma room temperature to 40° C. within about four minutes. Incidentally,temperature range that can be set by the temperature control part 6 isnot limited thereto.

Alternatively, the temperature of the separation column 3 is set ormodulated by the temperature control part 6 so that suitable temperaturemodulation is performed for adsorbing a specific substance in ameasuring sample onto the polymer layer surface of thetemperature-responsible gel modification column and, after a certainperiod of time, releasing the specific substance by modulating thetemperature to change the characteristics of the polymer layer surfaceof the temperature-responsible gel modification column.

FIG. 2 shows retention times of barbital-based drugs in the case wherethe temperature of the separation column 3 is modulated. The surface ofthe polymer layer of the temperature-responsible gel modification columnis changed from hydrophobic nature to hydrophilic nature by changing thetemperature of the separation column 3 from a high temperature to a lowtemperature at an arbitrary time and thereby it is possible to changethe retention time(s) of the objective component(s) to achieveseparation and fractionation.

The separation column 4 is a reversed phase separation column that isemployed in a reversed phase chromatography in which an octadecylsilyl(ODS) group is used as a representative.

The eluents B (12 a and 13 a) to be fed to the separation column 4 maycontain the eluent A 11 a or the eluent A 11 a and an organic solvent(methanol, acetonitrile, tetrahydrofuran, or the like to be commonlyused in a liquid chromatograph) contained therein. Moreover, the eluentsB (12 a and 13 a) to be fed to the separation column 4 may not have aconstant composition, and gradient elution may be performed in which thecomposition is changed, depending on the analysis time.

The reversed phase column achieves separation by hydrophobic interactionbetween an objective component and an eluent. More specifically, theelution time of the objective component is shortened when the ratio ofthe organic solvent in the eluent increases, while the objectivecomponent remains in the reversed phase column and is concentrated whenthe ratio of the organic solvent in the eluent decreases.

The separation column 4 is connected to the six-way valve 10 and thecomponents eluted from the separation column 3 are supplied togetherwith the eluent A 11 a at regular intervals.

The eluent A 11 a to be fed to the separation column 3 is an aqueousmobile phase.

The components supplied to the separation column 4 remain in theseparation column 4 and are concentrated since the eluent A 11 a is anaqueous mobile phase.

After the components eluted from the separation column 3 are supplied tothe separation column 4 together with the eluent A 11 a, the six-wayvalve 10 is switched and the eluents B (12 a and 13 a) are supplied tothe separation column 4.

The eluents B (12 a and 13 a) contain an organic solvent. Since theobjective component(s) concentrated in the separation column 4 ispromptly eluted by the eluents B (12 a and 13 a) containing the organicsolvent after the switching of the six-way valve 10, the peak(s) to bedetected becomes sharper.

FIG. 3 is a chromatogram showing a concentration effect by a liquidchromatographic analyzer. The peak separated in the first-dimensionalseparation column becomes sharper in the second-dimensional separationcolumn owing to the concentration effect.

On the other hand, the eluent A 11 a is an aqueous solvent having aconstant composition and the eluents B (12 a and 13 a) are solvents ofthe eluent A 11 a or the eluent A 11 a and an organic solvent containedtherein. The eluent A 11 a does not precipitate a salt in a piping orthe separation column 4 of the liquid chromatographic analyzer when theeluent comes into contact with the eluents B (12 a and 13 a) after theswitching of the six-way valve 10.

FIGS. 4A and 4B are chromatograms obtained by analyzing a serumcontaining phenobarbital, cyclobarbital, and pentobarbital as a sampleby the liquid chromatographic analyzer and analytical method shown inExample. As for the sample directly injected, proteins that arecontaminants are removed in the first dimension and clear separation ofthe drugs is performed in the second dimension.

Measurement Conditions

Sample: A tetrahydrofuran solution of phenobarbital, cyclobarbital, andpentobarbital that are psychoactive drugs was used as an authenticsample.

The authentic sample was dried to solidness under nitrogen andre-dissolved in a freeze-dry pooled serum and the resulting one was usedas a sample.

<First Dimension>

Pre-treatment column: P(NIPAAm-co-BMA 5%) gel modification column (4.6mm I.D.×150 mm L)

Mobile phase: 10 mM ammonium acetate (pH 6.5)

Flow rate: 1.0 mL/min

Detection wavelength: 220 nm

Injection amount: 10 μL

<Second Dimension>

-   -   Separation column: LaChrom IIC18 (5 μm) (4.6 mm I.D.×150 mm L)    -   Mobile phase: acetonitrile/10 mM ammonium acetate (pH 6.5)=75/25        (v/v)    -   Flow rate: 1.0 mL/min    -   Detection wavelength: 200 to 400 nm    -   Switching time: 5.5 to 9.2 min

In the disclosure, a temperature-responsible gel modification column isused as a separation column 3 but the separation column is not limitedthereto. The other portions are also not limited to the aforementionedembodiments and can be appropriately modified or improved. The material,shape, size, numerical values, form, number, arrangement, and the likeof each constituting element in the aforementioned embodiments arearbitrary and are not limited as far as they can achieve the invention.

What is claimed is:
 1. A two-dimensional liquid chromatographic analyzercomprising: a liquid feed part configured to feed a mobile phase; aninjection part configured to inject a sample; a first separation columnconfigured to separate and fractionate the sample containing a pluralityof components; a second separation column configured to separate thefractionated components; a first temperature control part configured tocontrol temperature of the first separation column; a second temperaturecontrol part configured to control temperature of the second separationcolumn; a detection part configured to detect the separated components,wherein the first temperature control part comprises a holder made ofmetal and configured to hold the first separation column so as totransmit a temperature change of the first temperature control part tothe first separation column.
 2. The two-dimensional liquidchromatographic analyzer according to claim 1, wherein the firsttemperature control part is configured to change the temperature of thefirst separation column from a first temperature to a second temperaturein a single-step manner and switch the temperature of the firstseparation column at a high speed.
 3. The two-dimensional liquidchromatographic analyzer according to claim 1, wherein the firstseparation column is a temperature-responsible gel modification columnhaving a functional polymer layer whose surface nature is reversiblychanged between hydrophilic nature and hydrophobic nature by temperaturemodulation or a reversed phase column configured to feed a 100% aqueousmobile phase, and wherein the second separation column is the reversedphase column.
 4. The two-dimensional liquid chromatographic analyzeraccording to claim 1, wherein the first separation column is atemperature-responsible gel modification separation column having afunctional polymer layer whose surface nature is reversibly changedbetween hydrophilic nature and hydrophobic nature by temperaturemodulation.
 5. The two-dimensional liquid chromatographic analyzeraccording to claim 1, wherein the mobile phase to be fed to the firstseparation column is an aqueous mobile phase, and wherein thecomposition of the mobile phase to be fed to the first separation columnis not changed at the separation and fractionation of objectivecomponent.
 6. The two-dimensional liquid chromatographic analyzeraccording to claim 1, wherein a temperature-responsible gel modificationcolumn is used as the first separation column, and a reversed phasecolumn such as an octadecylsilyl (ODS) column is used as the secondseparation column.
 7. A two-dimensional liquid chromatographicanalytical method comprising: feeding a mobile phase to a firstseparation column; injecting a sample containing a plurality ofcomponents to the first separation column; separating and fractionatingthe sample by using the first separation column; separating thefractionated components by using a second separation column; anddetecting the separated components, wherein the mobile phase to be fedto the first separation column is an aqueous mobile phase, and whereinthe composition of the mobile phase to be fed to the first separationcolumn is not changed at the separation and fractionation of objectivecomponent.
 8. The two-dimensional liquid chromatographic analyticalmethod according to claim 7, wherein a temperature-responsible gelmodification column is used as the first separation column, and areversed phase column such as an octadecylsilyl (ODS) column is used asthe second separation column.
 9. The two-dimensional liquidchromatographic analytical method according to claim 7, furthercomprising controlling the temperature of the first separation column toa target temperature.
 10. The two-dimensional liquid chromatographicanalytical method according to claim 7, wherein the sample is abiological sample, and wherein the separation and analysis of thecomponents in the biological sample are performed using thetwo-dimensional liquid chromatographic analyzer according to claim 1.